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BLISTER CANKER OF APPLE TREES 

A PHYSIOLOGICAL AND 

CHEMICAL STUDY 



A DISSERTATION 

SUBMITTED TO THE FACULTY 

OF THE OGDEN GRADUATE SCHOOL OF SCIENCE 

IN CANDIDACY FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

DEPARTMENT OF BOTANY 



BY 

DEAN HUMBOLDT ROSE 



Private Edition, Distributed By 

THE UNIVERSITY OF CHICAGO LIBRARIES 

CHICAGO, ILLINOIS 



Reprinted from 

The Botanical Gazette, Vol. LXVII, No. 2 

February 19 19 



TLhc mnivcxsit>s of CbicaGO 



BLISTER CANKER OF APPLE TREES; 

A PHYSIOLOGICAL AND 

CHEMICAL STUDY 



A DISSERTATION 

SUBMITTED TO THE FACULTY 

OF THE OGDEN GRADUATE SCHOOL OF SCIENCE 

IN CANDIDACY FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

DEPARTMENT OF BOTANY 



BY 

DEAN HUMBOLDT ROSE 



Private Edition, Distributed By 

THE UNIVERSITY OF CHICAGO LIBRARIES 

CHICAGO, ILLINOIS 



Reprinted from 

The Botanical Gazette, Vol. LXVII, No. 2 

February 19 19 






Qirt 
MAR O /SI9 



VOLUME LXVII NUMBER 2 



THE 

Botanical Gazette 



FEBRUARY igig 

BLISTER CANKER OF APPLE TREES; A PHYSIOLOGI- 
CAL AND CHEMICAL STUDY 

CONTRIBUTIONS FROM THE HULL BOTANICAL LABORATORY 246 

Dean H. Rose 

(with ten figures) 
Introduction 

It is now generally recognized that among the most important 
problems of plant pathology are those connected with the physiol- 
ogy of diseases whose etiology is already known. It is also recog- 
nized that this must be the physiology of the host, of the parasite, 
and of the two in relation to each other, and, further, that such a 
comprehensive view of all the factors involved furnishes the only 
rational approach to an understanding of the principles underlying 
immunity and disease resistance. 

In the present paper are given the results of a physiological 
study of the destructive disease known as Illinois or blister canker, 
the etiology of which, including the identity of the causal organism, 
Nummularia discreta (Schw.) TuL, was worked out by Hassel- 
BRiNG (22) in 1902. The work reported here is a continuation of 
an earlier investigation by the writer (30) on the oxidase activity 
of healthy and diseased bark; in addition there is included an 
account of the catalase activity and microchemical and macro- 
chemical analyses of both kinds of tissues. Further work is planned 
on the chemistry of the disease, on the role of other enzymes than 
oxidases, and on the physiology of the fungus itself in pure culture. 

105 



lo6 BOTANICAL GAZETTE [February 

The work was done in part at the Missouri State Fruit Experi- 
ment Station and in part in the Botany Department of the Uni- 
versity of Chicago. 

Historical 

The problem of oxidation by plant and animal tissues or tissue 
extracts has been studied by many investigators since the time of 
the pioneer work by Schonbein, the discoverer of ozone. An 
immense literature has accumulated, for reviews of which the reader 
is referred to pubHcations by Clark (14), Kastle (24), Battelli 
and Stern (5), and Atkins (3). In this paper only those articles 
will be cited which bear directly on the problem in hand. 

That pathological conditions in plants are often accompanied 
by increased oxidase activity has been shown repeatedly in recent 
years. Woods (35) found greater oxidizing power in the chlorotic 
portions of tobacco leaves affected with mosaic than in the green 
portions; this has been confirmed by Allard (i) and by Frei- 
berg (20). SoRAUER (31, 32) and Doby (17), working with leaf- 
roll of potatoes, found oxidase activity greater in diseased tubers 
than in healthy ones, although the former makes the point that 
this greater enzyme activity is to be considered a symptom of the 
disease rather than the cause. Bunzell (ii), working with the 
curly-dwarf disease of potatoes, showed by an extensive series of 
tests that "affected plants have a greater oxidase activity than 
healthy ones of the same age, both in the juice of their tubers and 
in the juice of their foliage." Similar results were obtained by 
Bunzell (id) in work with curly- top of sugar beets. All 4 of 
these diseases are of the so-called physiological type, and the 
question is still unsettled for the last 3 whether the increased 
oxidase activity is the cause of the disease or merely the result of 
disturbances due to the real but at present unknown cause. 

In the case of diseases whose cause is known the oxidase situa- 
tion seems to be about the same as for those already mentioned. 
Reed (29) found that the juice of apples affected with bitter rot 
{Glomerella cingulata) has greater oxidase activity than that of 
sound apples. In his previous work the writer (30) found that 
diseased apple bark shows greater oxidase activity than healthy 
bark, and is at the same time less acid. This seems to indicate 



19 ig] ROSE— BLISTER CANKER 107 

that the oxidizing power of a tissue bears some relation to its acidity, 
a relation which was rendered more probable by the fact that, 
according to titration and indicator tests, the acidity rises in the 
Bunzell apparatus during the course of an experiment at the same 
time that oxidation gradually decreases and finally ceases. The 
suggestion was made, therefore, that "the gradual slowing down 
of oxidation in the Bunzell apparatus is brought about in part by 
the accumulation of oxidation products, probably acetic and oxalic 
acids in the case of pyrogallol, and not by a using up of the oxidase 
through chemical combination between oxidase and oxidizable 
substance." The validity of this theory in the light of later investi- 
gation will be discussed in the experimental part of this paper. 

Experimental 

OXIDASE ACTIVITY 

Extracts of fresh bark. — An account will first be given of 
that part of the work done at the Missouri State Fruit Experiment 
Station. Extracts of fresh Ben Davis bark were used, prepared as 
follows: limbs were brought in from the orchard, the bark quickly 
ground in a meat grinder, and water and toluol added in the propor- 
tion of 4.25 cc. of toluol for each 100 cc. of water. The mixture 
was then allowed to extract at 28-30° C. for i hour, with frequent 
stirring, and filtered through filter paper. The proportions of 
water and toluol used, assuming that the fresh bark contained 
50 per cent water, were such as to make the extracts very nearly 
equivalent to those prepared for the earlier work (30) with dried 
bark. All data were corrected to the basis of dry weight deter- 
mined by weighing and drying samples of the ground bark in 
triplicate to constant weight in a bath at 95-99° C. 

Measurement of the amount of oxidation was made by means 
of the simplified Bunzell apparatus, using i cc. of the extract pre- 
pared as just described, and either 4 cc. of a i per cent solution of 
pyrogallol, 0.04 gm. of benzidine, or 2 drops (0.025 gm.) of 
guaiacol; water was added to make the final volume 6 cc. The 
various combinations of bark, oxidase reagent, and water were 
run in duplicate. 



io8 



BOTANICAL GAZETTE 



[FEBRUARY 



After the experiment had been set up in the incubator, i hour 
was allowed for the apparatus and solutions to come to a constant 
temperature. The manometers were then closed and the solutions 
mixed. No shaking machine was used, but the apparatus holder 
was tipped back and forth several times whenever a reading was 
taken. Allowance for temperature variations was made by run- 
ning with each experiment a blank containing only water and cor- 
recting the others by it. 

Table I gives the results of two representative experiments, 
showing the amount of oxidation of the 3 different reagents by 

TABLE I 

Oxidation of pyrogallol, benzidine, and guaiacol by extracts of healthy 

AND diseased BARK; MANOMETER READINGS CORRECTED AGAINST APPARATUS 
CONTAINING ONLY WATER; TEMPERATUTIE 28-31° C. 





Extract of fresh bark 


Extract of dried 

BARK 


Day of 


Pyrogallol 


Benzidine 


Guaiacol 


Pyrogallol 




Healthy 


Diseased 


Healthy 


Diseased 


Healthy 


Diseased 


Healthy 


Diseased 




Sample 38a 


Sample 386 


Sample 410 


Sample 41ft 


Sample 380 


Sample 386 






I 

2 

3 

4 

5 

6 


0.0 

0.71 

1.38 

1.70 

1.83 


0.0 

1.62 

2.46 

2.50 

2.50 


0.0 

0.17 

0.22 

0.41 

0.50 


0.0 
0.82 
I .21 
1.76 
2.07 


0.0 
0.02 
0. II 
0. 21 
0.27 


0.0 

0-43 
1. 16 

1.38 

I-5I 


0.0 
0.26 

0.53 
0.77 
0.86 
1.07 


0.0 

0.57 
0.94 
1.20 
1.36 
I-50 


7 

8 


2.12 
2.20 
2.36 
2.44 
1. 00 t 


2.66 

2.73 

2.86 

2.96 

I. 21 


0.60 
0.74 
0.77 
0.88 
1. 00 t 


2.31 

2.52 
2.67 

2-93 
3-32 


0.32 
0.32 

0-3S 
0.42 
1 .00 t 


1.68 
1. 91 
1.98 
2.18 
5.19 






9 

10 

Ratio . . 






i-Si 
1. 00 t 


1.94 
1.28 



extracts of both healthy and diseased bark. There are included 
also data from the earlier paper showing the amount of oxidation 
of pyrogallol by extract of dried bark. The results indicate that 
for approximately equal amounts of dry matter the dried bark is 
considerably less active than the fresh (fig. i). The decrease is 
probably due to the drying; this is shown more definitely by data 
to be presented later. It is to be noted that the oxidase activity 
of diseased bark is definitely greater than that of healthy bark, 



igig] 



ROSE— BLISTER CANKER 



109 



although the ratio between the two is greater where benzidine or 
guaiacol was used as oxidase reagent than where pyrogallol was 
used. The writer prefers to follow Bunzell in using the term 
oxidase activity or oxidizing power rather than "oxidase." Where 
the latter term occurs in this paper, it is used only for the sake of 
brevity, with no intent to imply any fixed notion as to the nature of 
the agent which brings about the oxidation. 

Titration and indicator tests on extracts of fresh bark showed 
the healthy bark to be more acid than the diseased, exactly as 
had been shown previously in the work with dried bark. No data 




Fig. I. — Oxidation of pyrogallol, guaiacol, and benzidine by extract of fresh 
bark, healthy and diseased, and extract of dried bark, healthy and diseased: A, 
pyrogallol and fresh healthy bark; B, pyrogallol and fresh diseased bark; C, ben- 
zidine and fresh healthy bark; D, benzidine and fresh diseased bark; E, guaiacol 
and fresh healthy bark; F, guaiacol and fresh diseased bark; G, pyrogallol and dried 
healthy bark; H, pyrogallol and dried diseased bark; //■ = healthy, Z) = diseased. 



are given, since the true condition, at least for dried bark, was 
determined more accurately by means of a potentiometer. 

Extracts of dried bark. — For the work at the University 
of Chicago bark was used which had been dried at 35-40° C. for 
2-3 hours, ground fine enough to go through a 40-mesh sieve, and 
stored air dry in zinc-capped Mason jars. A few of the experiments 
were run with oxidases precipitated from an extract of this bark 
powder, but in most of them the powder itself was used, o. 10 gm. 
in each apparatus. The reagents tested were pyrogallol and pyro- 
catechin, 4 cc. of a i per cent solution; benzidine 0.05 gm.; 



no BOTANICAL GAZETTE [February 

guaiacol 2 drops (0.025 g^''.)- Tests for any given set of condi- 
tions were always run in duplicate, sometimes in triplicate, or even 
quadruplicate. All experiments were shaken for 3 hours at the 
rate of 106 complete excursions per minute in a constant tempera- 
ture chamber provided with a fan driven from the outside, and 
then allowed to stand for 10-90 hours. Temperature variations 
were rarely greater than o . 5° during the shaking period, but some- 
times amounted to as much as 1.0° afterward, owing to less perfect 
control when the machinery was not in motion. Corrections for 
temperature variations were made as before by comparison with 
a blank containing only water. 

Potentiometer measurements were made with a hydrogen 
electrode like that described by Bovie (8), streaming hydrogen, 
3 resistance boxes as described by Michaelis (25, p. 131), a 
saturated calomel electrode, a normal element checked against 
another which had been calibrated by the United States Bureau 
of Standards, and a Leeds and Northrup dead-beat galvanometer. 
Hydrogen of high purity from a tank of the compressed gas was 
run through an electrically heated combustion tube containing 
platinized asbestos and then through the hydrogen electrode tube. 
The latter, together with the capillary from the calomel electrode, 
projected through a rubber stopper into the vessel containing the 
solution to be tested. Escape of hydrogen was provided for by a 
third opening in the stopper. An error was undoubtedly intro- 
duced here, due to displacement of CO2 from the solution, in 
cases where the hydrogen ion concentration was less than lO"^ 
(Michaelis, pp. 142-144), but since the only solutions showing 
this slight degree of acidity were mixtures of bark, water, and pyro- 
gallol for determination of hydrogen ion concentration before any 
oxidation had taken place, and since all others were found to be 
more acid, the error is probably negligible. It could have been 
avoided entirely by using a Hasselbalch shaking electrode had it 
and the time for using it been available. 

Among the hrst experiments run was one designed to test fully 
the oxidase activity of healthy and diseased bark when pyrogallol 
was used as the oxidizing substance. The results, given in table II, 
are the average of 5 closely agreeing determinations. These results 



I9I9] 



ROSE— BUSTER CA NKER 



III 



agree well with those obtained without a shaking machine in show- 
ing considerably greater oxidation by diseased than by healthy 
bark. The ratio between the two, 1.00:2.19, is larger than that 
found previously (1.00:1.28), the difference probably being due 
to differences in drying or possibly to the shaking itself. 

T-VBLK II 
Oxidation of pyrogallol by healthy and diseased apple bark; 

SAMPLES 3 and 4; TEMPER.A.TURE 27C±I.7°C. 



Time of 

RE.\DING 


1 

M.\NOMETER RE.'VDINGS, EX- 
PRESSED IN CM. OF MERCURY, 
CORRECTED AGAINST BLANK 
CONTAINING ONLY WATER 


1 Manometer readings, ex- 
pressed IN CM. OF MERCURY, 
TiMF OP CORRECTED AGAINST BLANK 

1 CONTAINING ONLY W.\TER 
READING 




Healthy Diseased 


, Healthy ' Diseased 


March 19 

2:45 I'-M 

3 : 00 

3:15 

3:30 

3:45 

4:00 

4:1s 

4:30 


0.0 

0.0 

O.IO 

0.16 

0.23 

0.31 

0.41 

0.45 


i 

0.0 

0.23 

0.48 

0.65 

0.80 

0.92 

I 05 
I. II 


March 19 0.48 i .25 
4:45 P.M.... 0.53 1.33 

5:00 0.61 1 .45 

5:15 0-64 I -57 

S : 30 0.59 1 . 49 

5:55 

March 20 ^ i . 10 2.41 
8:30 A.M....' 



In table III are summarized the results of an experiment to 
test the oxidizing power of both diseased and healthy bark on 
pyrocatechin, guaiacol, and benzidine. 

A comparison of the figures in table III with those in tables I 
and II shows that diseased bark causes greater oxidation of pyro- 
gallol, pyrocatechin, benzidine, and guaiacol than does healthy 
bark, and that both tissues cause greater oxidation of the first 
two reagents than of the last two. It is further shown by tables I 
and III that the amount of oxidation increases slowly for several 
days; in fact table III shows that it is practically doubled for all 
the combinations, except those containing pyrocatechin, during 
the 64-hour period following the 3 hours' shaking. This fact of 
an increase of oxidation on standing was observed to a greater or 
less degree with most of the bark material used in this work, and 
is in direct contradiction to Bunzell's explicit and repeated 
statement that oxidation in his apparatus comes to a definite end 
after 3 or 4 hours' shaking. The only exceptions the writer has 



BOTANICAL GAZETTE 



FEBRUARY 



noted were in those cases where the bark powder showed low 
oxidase activity to begin with, possibly due to injury of the "oxi- 
dase" during drying. 

TABLE III 

Oxidation of pyrocatechin, guaiacol, and benzidine by healthy and 

DISEASED bark; TEMPERATURE 29.4-29.7° C. 





Healthy 


Diseased 


Time of re.ading 


Benzidine 


Guaiacol 


Pyrocate- 
chin 


Benzidine 


GuaiaccJl 


Pyrocate- 
chin 


June 8, 1:30 P.M.. . . 

4:30 after 

shaking 3 hours . . 

June 9, 8: 10 a.m. . . 

2: 20 P.M. . . 

" 10, 9: 15 A.M. . . 

" II, 8:20 A.M. . . 


0.0 

0.08 
0.25 
0.38. 
0.40 
0.65 


0.0 

0-33 
0.35 
o.4§ 
0-5S 
0.65 


0.0 

113 
1-45 
i.6s 
1.85 
2. 12 


0.0 

0.65 
0.80 
0.98 
1.27 
1-45 


0.0 

0.7s 
1. 00 
1.07 
1.20 
1-47 


0.0 

3-77 
4-35 
4SS 
4.87 

5-12 



That the rate and temperature of drying have an effect on the 
oxidase activity as well as on the hydrogen ion concentration is 
clearly shown in table IV. 

TABLE IV 

Effect of rate and temperature of drying upon oxidase activity and 
hydrogen ion concentration of healthy and diseased apple bark 





Oxidation 






Temperature 


Degree of 
browning 


Sample 


After shaking 
3 hours 


After stand- 
ing 10 hours 


After stand- 
ing 15 hours 


Ph 


AND DURATION 
OF DRYING 


4 diseased . . 
6 " 


1.49 

2.25 

■ 1.58 

0.59 

1.07 

0.35 
0.62 


2.33 
2.42 
I .60 
0.80 
I. 12 

0.35 
0.72 


2.78 


5 
5 
5 
5 
S 
5 
4 


61* 

45 
16 

15 
04 
00 

80 


40°, 2 hours 
40°, 2 
40°, 4 
40°, 2 
40°, 2 
50:, 2 
35 ,4 


Slight 
Slight 
Much 


2 " 




3 healthy. . . 
5 " ... 
sa " . . . 
I " . . . 


1.23 


Very little 
Slight 
Very little 
Much 















* This figure is the negative logarithmic exponent of 10 where the whole expression 10='" is a 
measure of the hydrogen ion concentration in the solution. The larger it is, therefore, the smaller the 
hydrogen ion concentration it expresses. In this particular case it can be written 2.454X10"'' (6.00 — 
5.61 =o.3Q. Antilog 0.39 = 2.454). In the amplified form this becomes 0.000002454 (normal). 

Samples i, 2, 5, 5a, and 6 were all run in one experiment. 
Oxidation data for samples 3 and 4 are taken from table II and 
from another experiment not recorded in this paper. Sariiples 5 
and 5a were parts of the same lot of ground bark but received 



igiQj 



ROSE— BLISTER CANKER 



113 



different treatments as shown. The results show that oxidase 
activity is much reduced by drying at 35-40° for 4 hours (sample i, 
healthy; sample 2, diseased), or at 50° for 2 hours (sample 5a, 
healthy) . 

Hydrogen ion concentration. — Hydrogen ion determinations 
on mixtures of bark and water and of bark, water, and pyrogallol, 
used in the same proportions as in the oxidase apparatus, showed 
that pyrogallol has no effect on the reaction. It was found pos- 
sible to get constant initial readings on all mixtures- containing 
healthy bark and pyrogallol in 30-45 minutes; the same period 
sufficed for mixtures containing diseased bark and pyrogallol after 
they had been shaken in the oxidase apparatus, but not for similar 
mixtures freshly made up and not shaken. In these cases the 
potential increased slowly for an hour or two from about Ph = 5 . 60 
to Ph = 5.40, but never reached the figure given by healthy bark. 

Culpepper, Foster, and Caldwell (16), working with normal 
and diseased Red Astrachan apples, state that when titrations were 
made on fruit pulp suspended in water "the diffusion of acids out 
of the tissues continues for many hours and at slower rates in 
diseased than in normal fruits," but in the light of the following 
results the writer is inclined to think this increase of acidity was 
due to oxidation going on in the solutions, and not to diffusion of 
acids out from the tissues. 

TABLE V 

Correlation between oxidase activity and hydrogen ion concentration 

of mixtures containing pyrogallol, water, and either healthy 

or diseased bark; temperature 29-30.5° c. 



Stage of experiment 


Healthy 


Diseased 


Oxidation ' Pg 


Oxidation 


' Ph 


Before shaking 


0.00 5.15 
82 


0.00 
2.28 

2-59 

4.07 
4.96 


5.61 


After shaking 3 hours 

After standing 15 hours. . . . 

" 48 " .... 
" 64 " .... 




i.io 4.82 


4.89 


2.90 4.29 


4.29 



Increase in hydrogen ion concentration during oxida- 
tion.- — Experiments designed to test more fully the theory that 
oxidation causes an increase in acidity are summarized in table V. 



114 



BOTANICAL GAZETTE 



[FEBRUARY 



It is clear from table V that oxidation in these mixtures is 
accompanied by a marked increase in hydrogen ion concentration, 
and the conclusion certainly seems justified that there is a causal 
relation between the two. It is also seen that when oxidation 
comes to an end, both mixtures have the same reaction, Ph = 4.29, 
a condition suggesting that at this point the hydrogen ion is the 
limiting factor. 

BuNZELL (12) and Reed (28) have studied the effect of hydrogen 
ion concentration on oxidation, but apparently neither of them has 
realized that it might increase during the oxidation process (30). 
They apparently assume that the hydrogen ion concentration 
established at the beginning of an experiment remains constant 
until the end, whereas the results given show that in these cases 
it increased as long as the oxidation continued. 

In order to discover, if possible, what relation exists between 

oxidation and hydrogen ion concentration in the oxidase apparatus, 

further experiments were tried with mixtures of bark, dry pyro- 

gallol, and, instead of water, 5 cc. of buffer solutions containing 

various amounts of N/io sodium acetate and either N/io or 

N/ioo acetic acid. The initial reactions of these mixtures (before 

shaking) and of the buffers alone are given in table VI and shown 

graphically in fig. 2. 

TABLE VI 

Reaction, Ph, of buffer solutions and mixtures of buffer solutions, 
bark, and pyrogallol 



Solution 



Buffer alone i 6.02 5.73 

Buiier and healthy 

bark and pyro- 

gallol 

Buffer and diseased 

bark and pyro- 

gallol 




5-41 I 5-17 
5-36 j S-iS 
SSo I S-3I 



5 


6 


7 


8 


4.80 


4-53 


4.21 


3-90 


4-85 


4-58 


4.24 


3-98 


5.00 


4.61 


4-39 


4.08 



3.61 
3.61 

3-73 



Graphs B and C in fig. 2 show that while diseased bark absorbs 
H+ ions to about the same extent as the healthy, the latter absorbs 
more 0H~ ions; that is, its titration acidity is greater, which 
is exactly the condition found by titration with N/20 sodium 
hydroxide (30). The Ph values at points where B and C cross A 



igiQ] 



ROSE— BLISTER CA NKER 



115 




v lcC-^b ACETIC ACID to \ CC-y(o SOD, ACETATE 



9K10.24 TJZ 2:56 1.28 0. 0.32 0.16 0.08 0.04 

Fig. 2. — F^ of mixtures of bark, pyrogallol, and various buffer solutions before 
and after oxidation had ceased: A, P^ of buffer solutions; B, P^ of mixtures of 
buffer solutions, pyrogallol, and diseased bark before oxidation; C, P^ of mixtures 
of buffer solutions, pyrogallol, and healthy bark before oxidation; D, P^ of mixtures 
of buffer solutions, pyrogallol, and diseased bark after oxidation; E, P^ of mixtures 
of buffer solutions, pyrogallol, and healthy bark after oxidation. *Only acetic acid 
used here. 



ii6 



EOT A NIC. 4 L GA ZE T TE 



FEBRUARY 



(healthy bark about 5.10, diseased about 5.65) agree well with 
those determined without the buffer (Ph healthy = 5.15, diseased = 
5.61); the latter are taken, therefore, to represent practically the 
actual acidity in each case. This is based on the assumption that 
if the acidity of a buffer solution is the same as that of a mixture 
of bark, pyrogallol, and water, no change in acidity will take place 
when the buffer is used instead of water. 

Effect of buffer solutions. — The oxidations brought about 
by mixtures of bark, pyrogallol, and the various buffer solutions 
are given in table VII, together with the initial Ph of these mixtures 
and their Ph after oxidation had practically ceased. 

TABLE VII 

Oxidation by mixtures of bark and pyrogallol with various buffer 
solutions; temperature 29-30° c. 





Healthy 


Diseased 


SOLUTION 


Oxidation 


Initial Pg 


Final Pg 


0.xidation 


Initial P^ 


Final P^ 


I 




t: en 




4.68 

4.78 ■ 


5-76 
5-70 
550 
531 
5.00 
4.61 

4-39 
4.08 

3-73 
5.61 


485 
485 


2 


1.68 


5 
5 
S 
4 
4 
4 
3 
3 
S 


52 
36 
IS 
85 
58 
24 
98 
61 
15 


4.58 


3 


4 

e 


2.15 


4-34 


4 36 


4-7S 


6 

7 

8 


1-95 
1.80 

1-55 
0-53 
2.22 


398 
3.65 
356 
3-35 
4.29 


4.48 
4.12 


4.60 
425 


9 

Check 


1.82 
4-27 


3-68 
4.29 



The principal fact shown by the results in table VII is that the 
Ph (4 29) reached by mixtures of pyrogallol, water, and either 
healthy or diseased bark when oxidation comes to an end is not 
sufficient to inhibit oxidation when the mixture has that Ph value 
to begin with; in fact, a greater degree of acidity does not inhibit 
entirely, since a healthy bark mixture with an initial Ph of 3.61 
gave an oxidation (a mercury rise) of 0.53 cm., and a diseased 
bark mixture with an initial Ph of 3.78 gave an oxidation of 
1.82 cm. The check, bark, pyrogallol, and water gave, in the 
former case, 2.22 cm. mercury rise, and in the latter 4.27 cm. 

It might seem from this that the acidity brought about in 
mixtures of bark, pyrogallol, and water is not the factor which 



1919] ROSE— BLISTER CANKER 117 

brings oxidation to an end. It seems more reasonable to suppose, 
however, that the time factor is of importance here; that is, that 
an acidity of Ph = 4.29 is more effective when reached gradually 
than when established as a starting point. Looking at the situa- 
tion from another angle, we may say that inhibition is total if the 
initial hydrogen ion concentration is high enough, but will be only 
partial if the concentration is lower; but since partial inhibition 
means some oxidation, which in itself increases acidity, the process 
in time necessarily comes to an end. The hydrogen ion concen- 
tration at that point will depend on what it was in the beginning, 
but will never be equal to that which causes total inhibition. 

That this theory fits the facts is shown by table VII. Oxidation 
took place in all the mixtures, the amount depending on the initial 
hydrogen ion concentration, except where diseased bark was used 
with buffer no. 4. Acidity increased in all the mixtures but one, 
diseased bark with buff'er no. 6 (see tables VI and VII). The 
increase in acidity is shown graphically in fig. 2. It is unexpectedly 
small for diseased bark except where the 3 most alkaHne buffers 
were used, a condition which suggests the need of further experi- 
ments. 

In figs. 3 and 4 are shown graphically the oxidation data given in 
table VII, representing the final amounts of oxidation for each set 
of tests (healthy and diseased bark with the different buffer solu- 
tions) . In addition there are shown graphs for several earlier stages 
in each experiment. These graphs show that below i X iC^ (p^ = 4) 
for healthy bark, and 2.5X10"^ (Ph = 4.39) for diseased bark, 
oxidation drops rapidly as acidity increases. Above these points 
the changes are not so marked. The hydrogen ion concentration 
for total inhibition, estimated by extrapolation to the base line, 
lies between 3.55 and 3.80X10"-* for healthy and between 3.55 
and 4 . 27 X io~'* for diseased bark. All these figures closely approxi- 
mate those found by Bunzell (12) for potato oxidase, 2 . i- 
2.8Xio~^, and by Reed (28) for apple oxidase, 5 .0-7 .cXio"'*. 

The results given in table VII show that hydrogen ion con- 
centration is not the only factor effective in controlling oxidation 
in the apparatus, and consequently that the lower hydrogen ion 
concentration of diseased bark cannot account entirely for its 



ii8 



BOTANICAL GAZETTE 



[FEBRUARY 



greater oxidizing power. For example, when both kinds of bark 
were brought to approximately the same hydrogen ion concentra- 
tion by buffer no. 6, the final amount of oxidation (mercury rise) 
for healthy bark was i .95 and for diseased 4.48, the filial Ph 3 .98 
and 4 . 60 respectively. The total oxidase activity of the diseased 
plant is the joint oxidase activity of the host and parasite, while 



o 



i 9 


8 


7 


6 


4 


2 


^ 3.61 


3.98 

1 


4.24 


4.58 


5.15 


5.52 

— C 

_-j B 

1 

1 ■ 


// 


^ 1 


A 


/ 


' 


1 Ph 




1 


1 
1 



Fig. 3. — Oxidation of mixtures of healthy bark, pyrogallol, and various buffer 
solutions: A, after 3 hours; B, after 22 hours (19 hours without shaking); C, after 
29 hours; D, after 48 hours; ^, bark, pyrogallol, and buffer solutions as indicated by 
numbers; B, initial P^; points of plotting marked by vertical broken lines. 

the oxidase activity of the healthy plant is that of the host alone. 
This may account in part for the difference both in rate of activity 
and in the Ph concentration at the time the action ceases. 

Nature of equilibrium reached. — Bunzell (13), in experi- 
ments with potato peel powder, has obtained what he considers 
evidence that "the activity of the plant powder is not paralyzed 
by the products formed in the course of the reaction." He found 



igig] 



ROSE— BLISTER CANKER 



119 




Fig. 4. — Oxidation by mixtures of diseased bark, pyrogallol, and various buffer 
solutions: A, after 23 hours (shaken 2 hours of this time) ; B, after 45 . 5 hours; C, after 
69.5 hours; A and B as in fig. 3. 



BOTANICAL GAZETTE 



[FEBRUARY 



that by adding a second portion of the powder to the apparatus 
in which oxidation by the first portion had ceased he could cause 
a further increase in oxidation, the amount of increase varying 
with the oxidase reagent used. The writer has found a similar 
increase in oxidation when more oxidase reagent is added, after 
oxidation ceases. The results of an experiment of this kind are 
summarized in table VIII. Results are given beginning with the 

TABLE VIII 

StTMMARY OF RESULTS FROM AN EXPERIMENT TO TEST EFFECT OF ADDING 
FRESH SUPPLY OF OXIDASE REAGENT. 



Experiment 



Stage of experi- 
ment 



Increase in oxidation (cm. of 
mercury rise) 



Effect of adding 7 and 4 drops i per cent 
benzidine to apparatus 10 and 11 on 
ninth day, 8 and 9 as checks 

Effect of adding 10 drops i per cent ben- 
zidine to apparatus 10 and 11 on 
eleventh day, 8 and 9 as checks 

Total effect of i per cent benzidine, 8 as 
check 

Effect of adding 0.06 gm. of pyrogallol 
to 8 on twenty-sixth day 

8 as check 

Effect of adding 0.06 gm. benzidine to 9 
on fourteenth day, 8 as check 

Effect of adding 10 drops absolute 
alcohol to 9 on twenty-first day, 8 as 
check 



9th to nth I 0.00 



nth to 14th 

9th to 26th 

26th to 41st 
9th to 26th 

14th to 2ISt 
2ist to 26th 



0.17 
0.47 



1-53 
0.47 



o. 19 



0.23 



0.49 
1. 16 

1-95 



0.19 

0.71 
1-34 



0.74 
0.68 



ninth day of the experiment. Up to that time oxidation in all 
4 of the tubes was practically the same, the average being 3.12 
(cm. of mercury rise). Alcohol was used at the beginning of the 
experiment to discover whether it has an inhibiting effect on oxi- 
dation, and later, when solid benzidine was added, to bring the 
benzidine into solution more rapidly. The results show that, in 
the small quantities used, the alcohol had no inhibiting effect 
(table VIII, ninth day, apparatus 10 and 11), and probably did 
bring the benzidine into solution (twenty-first-forty-first day, 
apparatus 9). 

The most important fact shown by these results is that after 
oxidation had practically ended, the addition of more oxidase 



iqiq] rose— blister CANKER 121 

reagent was followed by a marked increase in oxidation. For 
example, in table VIII it is seen that from the ninth to the twenty- 
sixth day oxidation in apparatus 8, containing pyrogallol and bark 
extract, showed an increase of only o .47 cm., while tubes 10 and 11, 
also containing pyrogallol and bark extract to which benzidine 
solution was added later, showed an increase of i .95 and i .34 cm. 
respectively. Equally marked excess over the check was obtained 
when solid pyrogallol or solid benzidine was added. One might 
infer that the oxygen admitted, when the tubes were opened to 
introduce reagents, increased oxidation, but this effect could hardly 
account for the difference observed. Bunzell states that exhaus- 
tion of oxygen is not the limiting factor, and experiments by the 
writer have shown that, when a fresh oxygen supply is allowed to 
enter the apparatus, the subsequent increase in oxidation is small. 

The fact that after oxidation ends it can be started afresh 
by the addition of fresh plant material or of fresh oxidase reagent 
suggests that the equilibrium reached is a false one, like the third 
case described by Hober (23, p. 671), in which a reaction product 
of the catalytic reaction brings about equilibrium by an inactiva- 
tion of the catalyzer. A test for this condition according to Hober 
is that reaction begins again when more catalyzer is added, as in 
the case of the hydrolysis of amygdalin by emulsin. The similarity 
between the two reactions, however, does not prove that the oxida- 
tion catalyst is an enzyme, for it may be non-enzymic in nature 
and still be inactivated by the products of the catalytic reaction. 

An idea of the nature of the oxidase reaction was obtained by 
testing some of the data by the formula for unimolecular reaction, 

k= - log. . In these calculations the total amount of oxida- 

t a—x 

tion (mercury rise) at the end of the shaking period was assumed 
for the value of a, and the amount of oxidation at the end of each 
15-minute interval for the value of x. The figures which should 
be used, of course, are the total amount of pyrogallol at the begin- 
ning of the experiment and the amount oxidized at the end of each 
15-minute interval, but such figures would be difficult to obtain. 
The writer sees no reason why the values used for a and x do not 
truly represent the course of the reduction. 



122 



BOTANICAL GAZETTE 



[FEBRUARY 



In most cases the values of k given in these tables are fairly 
constant and may be considered a strong indication that the 
oxidase reaction is unimolecular. In table XI, column 3, table XII, 



TABLE IX 
Healthy bark and pyrogallol 



i (mill.) 


X (mercury rise 
in cm.) 


a—x 


, I , a 

k = - log. 

I a—x 


15 


0.14 
0. 19 
0.24 

0.34 
0.44 
0.49 
0-54 

0.63 
0.68 
0.74 
0.79 
0.86 


0.72 
0.67 
0.62 
0.52 
0.42 

0-37 
0.32 
0.23 
0.18 
0. 12 
0.07 


*, 


0.00514 
0.00361 
0.00315 
0.00365 
0.00415 
. 00407 
. 00409 
0.00477 
0.00503 

0. 00 c 70 


^0 


41; 


60 


71; 


no 


los 

120 


135 

150 


165 


0.00660 


180 




Mean .... 




.OOA2Z 













* Brackets in this and following tables indicate those values of k 
which were considered in calculating the mean. 



TABLE X 

Diseased bark and pyrogallol 



t (min.) 


X (mercury rise 
in cm.) 


a—x 


k=- log. 

t a—x 


15 


0.13 
0.30 
0.50 
0.65 
0.72 
0.85 
0.99 
1.04 
I IS 

1. 18 

1-25 

1.38 


1-25 

1.08 
0.88 

0.73 
0.66 

0.53 
0.39 
0.34 
0.23 

0.20 

0.13 


0.00286 


%o 




0.00355 
0.00434 
0.00461 


4? 


60 


7c 


0.00427 
0.00461 
. 005 2 2 
0.00507 
0.00576 
,0.00559 
O.O062T 


00 


ic; 


120 


I?C 


ISO 

leq 


180 




Mean. . . . 




. 00A78 













column I, and table XIV, column i, the values for k show a gradual 
increase throughout the experiment, and can scarcely be taken 
to indicate a unimolecular reaction. Table XII, column i, how- 
ever, is checked by tables IX and XII, column 3, the mean value 



iQIQ] 



ROSE— BLISTER CANKER 



123 



of k being nearly the same in all 3 cases, although it is doubtful 
whether a mean for table XII, column i, is really significant. 

TABLE XI 

Values of k calculated from data obtained in experi- 
ments WITH APPLE BARK, K2CO3, AND PYROGALLOL 





k 


/ (min) 


K^COj and 
pyrogallol 


K,C03 and 
pyrogallol 


Healthy bark, 
KjCOj, and 
pyrogallol 


Diseased bark, 
KCjOj, and 
pyrogallol 


15 

30 

45 

60 

75 

90 

105 

120 

135 

150 

165 

180 




0.00747 
0.00776 
0.00774 
0.00785 
. 00808 
0.00767 
0.00765 
0.00786 
0.00811 
0.00768 
. 00805 


< 


O.OD552 
. 00803 
0.00773 
0.00786 
0.00819 
0.00750 
0.00709 
0.00890 
. 00941 
. 00947 
0.00936 


: 


0.00114 
. 00380 
. 0043 I 
0.00368 
0.00472 
0.00492 
0.00514 
0.00470 
0.00633 
. 00663 
0.00692 




0.00525 
'0 . 00600 
0.00615 
. 00604 
. 00606 
0.00628 
0.00642 
0.00657 
. 00700 
,0.00678 
0.00862 


Mean. . 


0.00781 


. 00834 


0.00482 


0.00635 



TABLE XII 

Values of k calculated from data obtained in experi- 
ments WITH APPLE BARK, PYROGALLOL, AND PYROCATECHIN 



t (min.) 



IS- 
3°- 
45- 
60. 

75- 
90. 

loS- 
120. 

135- 
ISO. 
165. 
180. 



Mean. 



Healthy bark 
and pyrogallol 



O . 00246 
0.00277 
0.00322 
0.00383 
0.00493 
o . 00460 
0.00501 
0.00584 

0.00886 



o . 00430 



Diseased bark 
and pyrogallol 



0.00458 
0.00528 
0.00515 
0.005x5 
0.00510 
0.00530 
0.00507 
0.00575 
o . 00604 
o . 00744 



0.00527 



Healthy bark 

and pyrocat- 

echin 



0.00502 
0.00429 
0.00357 
o . 00346 
0.00416 
o . 00433 
0.00426 
o . 00434 
o . 00483 
0.00548 
0.00590 



0.00451 



Diseased bark 

and pyrocat- 

echin 



O . 00483 
O . 00494 
0.00510 
O . 00488 
0.00526 
0.00536 
0.00553 
0.00584 
0.00613 
o . 00690 
o . 00866 



0.00521 



Confirmation of the results with apple bark is found in 
table XIII and table XIV, column 2, based on data obtained by 



124 



BOTANICAL GAZETTE 



[febru.-vry 



BuNZELL (9, 13) with tulip tree leaves and with potatoes, although 
the mean value of k in all 3 cases is much larger than that found for 
bark. Attention has already been called to the fact that the data 
in table XIV, column i (also from Bunzell's work), fail to fit the 
equation for a unimolecular reaction. The fact of a marked rise 

TABLE XIII 

Values of k calculated from data published by 

BuNZELL (9) FOR POTATO JUICE 
AND PYROGALLOL 



{ (min) 



10. 
20. 
30. 
40. 
SO. 
60. 
70. 
80. 
90. 



k* 






Mean. 



0.0315 
0.0266 
0.0240 
0.0216 
0.0244 
0.0277 
0.0255 
0.0283 



0.0262 



/ (min) 



10. 
30. 
45- 
60. 

75- 
90. 

105 



Mean . 



*t 



0.0246 
0.0277 
0.0199 
0.0168 
0.0174 
0.0233 



0.0208 



.* 23, p. 29, table Vn, columns i and 4. 
t 23, p. 26, table II, columns 5 and 7. 



TABLE XIV 
Values of k calculated from data published by Bunzell (13) 





k 


t (min.) 


k 


/ (min.) 


Spinach leaves 
and para-cresol 


Tulip tree leaves 
and phlorliizin 


Spinach leaves 
and para-cresol 


Tulip tree leaves 
and phlorhizin 


15 

30 

45 

60 


0.00374 
0.00654 
. 00640 
. 00940 
0.01092 


0.0124 
0.0119 
0.0133 
0.0137 


90 

105 


O.OIO18 






120 






135 






75 


Mean 




0.0137 









in the value of k toward the end of the experiments with bark may- 
mean that at that point the "oxidase" oxidizes not constant frac- 
tions but constant weights of pyrogallol in a given time (Philip 27, 
p. 295). The data at hand, however, are insufficient for a veri- 
fication of this hypothesis. 



iqiq] 



ROSE— BLISTER CANKER 



125 



A unimolecular reaction is one in which the concentration of 
only one substance is changed. If oxidation of pyrogallol by plant 
material in the oxidase apparatus be such a reaction, the substance 
whose concentration is changed is pyrogallol. The "oxidase" then 
appears as the catalyst, its concentration remaining unchanged 
during the course of the reaction. Even at that it is not neces- 
sarily proved to be an enzyme, since the linear relationship between 
time and amount of change is also shown in the oxidation of pyro- 
gallol by potassium carbonate. 

Effect of adding protective colloids. — Bayliss (6) and 
Perrin (26) have suggested that the oxidizing enzyme is an active 
form of the colloidal hydroxide of manganese, iron, or copper, 
kept in this active state by an emulsion colloid such as gum or 
albumin, acting as a protective colloid. Tables XV and XVI 
show the effects of additions of gelatine and gum arable. Table XV 
shows that o . 2 per cent gelatine increases considerably the oxida- 
tion by healthy bark and only slightly that by diseased bark. 
Three other experiments with pyrogallol and 2 with pyrocatechin 
with o . 2 per cent gelatine added showed similar results. The use 
of o . 8 per cent gelatine with pyrogallol also showed a similar effect. 
Both 0.2 and 0.8 per cent gum arable had Httle or no effect on 
healthy bark and a slight accelerating effect on diseased bark. 

TABLE XV 

Effect of o . 2 per cent gelatine on oxidation of pyrogallol by healthy 

AND diseased BARK; TEMPERATURE 2 2-24° C. 



Time of reading 


Healthy 


Diseased 


Without gelatine 


With gelatine 


Without gelatine 


With gelatine 


May i8, 7:03 p.m 


0.0 

0.77 
0.94 
1-37 
i.6s 


0.0 

0.81 
1.32 
2.26 
2.74 


0.0 

2.22 
2.40 
2.88 

317 




10:03 P-^i- after 
shaking 3 hours . . 
" 19, 8: 10 A.M 


2.24 

2.54 
3.00 

3-27 


" 20. q: 'iK A.M 


" 21, 8: 15 A.M 





Since gelatine is amphoteric, one might infer that it or its 
splitting products act as buffers, thus reducing the rate of increase 
of the hydrogen ion concentration with progress of the oxidation 



126 



BOTANICAL GAZETTE 



FEBRUARY 



(fig. 5). Table XVII, however, shows that gelatine has little 
effect on the hydrogen ion concentration of oxidizing mixtures of 
either healthy or diseased bark. 

Precipitated oxidases. — Experiments were run using pre- 
cipitated "oxidases," prepared as follows: 2 gm. of bark were 
allowed to extract with 10 cc. of water and 5 drops of toluol for 
I hour; the extract was then squeezed through moist cheesecloth 
on to coarse filter paper, the beaker washed with five i cc. portions 
of water and the filter paper finally with two more; 50 cc. of 95 per 




Fig. 5. — Effect of 0.8 per cent gum arable and 0.8 per cent gelatine on oxidation 
of pyrogallol by healthy and diseased bark: A, healthy bark; B, healthy bark and 
gum arable; C, healthy bark and gelatine; D, diseased bark; E, diseased bark and 
gum arable; F, diseased bark and gelatine. 

cent alcohol were then added to the filtrate (concentration of alcohol 
about 70 per cent), the whole allowed to stand for 10 minutes and 
the flocculent precipitate collected on a hard filter by gentle suction 
with a filter pump; 150 cc. more alcohol were then added to the 
filtrate (concentration of alcohol now about 90 per cent) and the 
whole allowed to stand for i hour, since precipitation was slow, 
before collecting this second fraction on the filter with the first. 
The precipitate from diseased bark was much browner than that 
from healthy bark. Whether this bears any relation to its greater 
oxidase activity is not known. 



iqiq] 



ROSE—BLISTER CANKER 



127 



For tests in the oxidase apparatus the combined precipitates 
were dissolved in 20 cc. of water, and 2 cc. of this solution contain- 
ing the precipitate obtained from o. i gm. of bark was put in each 
apparatus together with the usual amounts of pyrogallol and water. 

TABLE XVI 

Effect of 0.2 per cent gum arabic, 0.8 per cent gum Arabic, and 0.8 per 

CENT gelatine ON OXIDATION OF PYROGALLOL BY HEALTHY AND 
DISEASED BARK; TEMPERATURE 21-23° C. 





Healthy 


Diseased 


Time of re.^ding 


No 
addition 


Gelatine 


Gum arabic 


No 
addition 


Gelatine 

0.8 per 

cent 


Gum arabic 




0.8 per 
cent 


. 2 per 
cent 


0.8 per 
cent 


. 2 per 
cent 


0.8 per 
cent 


At beginning. . . . 
After shaking 3 

hours 

After 18.5 hours 
After 42 hours. . 
Average of 


0.00 

0.70 
1.03 

2 


0.00 

0.78 

1-34 
2. 20 


0.00 

0.65 
1. 00 


0.00 

0.71 
1. 01 
1-54 


0.00 

1.88 
2.36 
2-73 

2 


0.00 

2.23 
2.69 
3-18 


0.00 

2.19 
2.71 


0.00 

2.05 
2.46 
2-95 

















In table XVIII are given results showing the oxidizing power of 
these solutions, with and without gelatine (fig. 6) . 

The relation observed with bark powder still holds here, that 
diseased material is more active than healthy. On the other hand, 
gelatine increases oxidation by the precipitate from extract of 

TABLE XVII 
Reaction of mixtures of bark and pyrogallol with gelatine (o . 2 per cent) 

AND without at VARIOUS STAGES OF OXIDATION PROCESS 



Time of reading 


Healthy 


Diseased 


Without gelatine 


With gelatine 


Without gelatine 


With gelatine 


[Initial 


S15 
4.82 
4.29 


5-15 
4.84 
4-35 


5.61 
4.89 
4.29 


5.60 
4.86 
452 


P ] After 15 hours 

= [After 64 hours 



diseased bark, but is without marked effect on that from healthy 
bark, the reverse of the condition found when bark powder was 
used. 

There were indications in the preliminary work that the alco- 
holic precipitate from bark extract was easily separated into 2 



128 



BOTANICAL GAZETTE 



FEBRUARY 



fractions, hence it seemed worth while to collect these separately. 
This was done for both healthy and diseased tissue and gave 

TABLE XVIII 

OxroATION OF PYROGALLOL BY AQUEOUS SOLUTIONS OF PRECIPITATED OXIDASE 

FROM HEALTHY AND DISEASED BARK, WITH AND WITHOUT 

GELATINE ; TEMPERATURE 29 . 3-3O . 3° C . 





Healthy 


Diseased 




Without gelatine 


With gelatine 


Without gelatine 


With gelatine 


June 28, 4: 35 P.M 


0.0 
0-31 

0.35 
0.42 


0.0 
0-33 

0.46 
0.46 


0.0 
0.68 

1 .01 
1.08 


0.0 


" 20, 8:4"; A.M 


0.72 

1.24 
1.56 


" "11:45 A.M. after 

shaking 3 hours . . . 
" "^o, 8: '?o A.M 






Fig. 6. — Oxidation of pyrogallol by precipitated oxidases from healthy and 
diseased bark, with and without gelatine, shaken only during period from A to B: 
A, precipitate from healthy bark without gelatine; B, precipitate from healthy bark 
with gelatine; C, precipitate from diseased bark without gelatine; D, precipitate 
from diseased bark with gelatine. 

precipitates whose air dry weights, determined by the use of tared 
filters, were as follows: 





From extract of 
healthy bark 


From extract of 
diseased bark 


Fraction i 

Fraction 2 


0.0099 gro- 

. 0080 


0.0532 gm. 
0.0164 


Total 


0.0179 


0.0696 





The greater amount of precipitate from diseased bark may or 
may not be directly connected with its greater oxidase activity. 



igig] 



ROSE— BUS TER CA NKER 



129 



Further study is necessary to show the facts. A test of these pre- 
cipitates with pyrocatechin showed that while the 2 fractions from 
healthy bark are about equal in oxidizing power the first fraction 
from diseased bark is 11 times as active as the second (fig. 7). 




Fig. 7.— Oxidation of pyrocatechin by precipitated oxidases from healthy bark, 
without gelatine: A, fraction i; B, fraction 2; C, fractions i and 2 tested together; 
D, sum of fractions i and 2 tested separately. 



Other precipitates were prepared using 25 cc. of alcohol for the 
first fraction and 100 cc. more for the second. The oxidase activity 
of these, tested separately and combined, with and without gelatine, 
is shown in table XIX. 

TABLE XIX 

Oxidase activity of first and second fractions from bark extract 
tested separately and combined; temperature 29.5-30.0° c. 





Without gelatine 


With gelatine 


Bark extract 


Sum of fractions 

I and 2 tested 

separately 


Fractions i and 2 
combined 


Sum of fractions r-, ,.- j 
I and 2 tested Factions i and 2 
separately , combmed 


Healthy, after 23 hours .... 
Diseased, "38 " . . . . 


0.65 
1.82 


0.84 
2.00 


0.76 0.84 
2 . 64 3 . 06 



The mechanism by wliich gelatine increases the oxidase activity 
is not clear. It is evidently not through buffer action, as shown by 
its lack of effect on the hydrogen ion concentration (table XVII, 
figs. 8, 9, 10). Special tests showed that there was no hydrolysis 
of the gelatine to amino acids, in either healthy or diseased bark, 
which would increase its buffer effect. If gelatine is effective 
through its action as a protective colloid, its effect in this direction 
must be very complex, as shown by its difference in effect on bark 
mixtures and precipitated oxidases. 



I30 



BOTANICAL GAZETTE 



FEBRUARY 





0) . 










1.2 








J» 


€| 


0.6 


^■^ 1 

ft) 1 1 


ziiz:^^^^ 


— ■ -^Z- ' 


_=ti— ^=^ 


B 



7/ me /n hours 



Fig. 8. — Oxidation of pyrocatechin by first and second fractions of healthy bark, 
with gelatine (for explanation of lettering see legend for fig. 7). 




12 3 16 40 

Fig. 9. — Oxidation of pyrocatechin by first and second fractions from diseased 
bark, without gelatine (for explanation of lettering see legend for fig. 7); points of 
plotting marked by vertical broken lines. 




12 3 16 40 

Fig. 10. — Oxidation of pyrocatechin by first and second fractions from diseased 
bark, with gelatine (for explanation of lettering see legend for fig. 7) ; points of plotting 
as in fig. 9. 



iqiq] • ROSE— BLISTER CANKER 131 

The difference in the effect of gelatine and of gum arabic on 
oxidation by healthy bark may depend on differences in the col- 
loidal solutions they form. An artificial oxidase prepared by 
Dony-Henault (18) from manganese formate, sodium bicarbonate, 
and gum arabic could be destroyed by heat; but one prepared by 
Trillat (33) from albumin and manganese could not be so 
destroyed. Bayliss (6, p. 585) thinks the difference here "clearly 
depends on the nature of the emulsion colloid in association with 
the metal." On the other hand, what little increase in oxidation 
gum arabic produces may be due to an oxidase naturally present in 
it (BouRQUELOT 7), although an experiment designed to test this 
question gave negative results. One per cent gum arabic plus 
I per cent pyrogallol, and pyrogallol alone, were placed in separate 
oxidase tubes and shaken twice during each 24 hours. At the end 
of 3 days the mercury rise was 0.32cm. in the first case and 
o . 20 cm. in the second, a difference almost within the limits of 
error in reading the manometers. 

The data given in table XIX show that when the precipitate 
is collected in 2 fractions, these fractions have a greater oxidase 
activity if combined than if used separately. This condition seems 
to be about the same as that described by Bach and Chodat (4) 
for Lactarius vellereus. They found that by the fractional pre- 
cipitation of an aqueous solution of the oxidase of this fungus, by 
alcohol, 2 fractions could be obtained possessing markedly different 
properties. The first of these was almost insoluble in 40 per cent 
alcohol and had the properties of a weak oxidase; the second was 
soluble in 40 per cent alcohol but insoluble in pure alcohol and had 
no oxidizing powers. This fraction, however, was found to impart 
greater activity to hydrogen peroxide as an oxidizing agent; it 
was also found to increase markedly the oxidizing powers of the 
first fraction. The chief difference between this situation and 
that found in the work with apple bark is that in the latter 
case the first fraction has more than a weak oxidase activity, 
while the second, possibly because of incomplete separation of 
the fractions, is not entirely without it. No tests have been 
made of the behavior of the second fraction toward hydrogen 
peroxide. 



132 BOTANICAL GAZETTE [February 

Oxidase activity of the fungus in pure culture. — A fungus 
powder was prepared according to the method employed by 
Reed (29) from mats of Num?Hularia mycehum grown in the 
potato extract medium described by Duggar (19). A test with 
3 Bunzell tubes using o . i gm. of fungus powder, 4 cc. of i per cent 
pyrogallol, and i cc. of water gave after 4 days an average mercury 
rise of 2.35 cm. Quantitative tests on the medium in which the 
fungus had grown showed "oxidase" present there also. From 
these results it appears probable that the greater oxidase activity 
of diseased bark is due to a summation of the oxidase activity of 
normal bark and of the canker fungus itself. This may also account 
for the difference in behavior of the oxidases of the two. 

The general conclusion to be drawn from the preceding data is 
that diseased bark has greater oxidase activity than healthy bark, 
probably because of lower acidity and greater degree of dispersion 
of the oxidizing agent, and because of an actually greater oxidase 
content. The lower tannin content of diseased bark (see macro- 
chemical work) may also be a contributing factor, since tannins 
are known to cause inhibition of oxidase action. This factor is 
probably eliminated when precipitated oxidases are used. 

In reference to the Bunzell apparatus it may be said that while 
it gives valuable comparative measurements of oxidase activity, 
those using it must realize its limitations Conditions within it 
are artificial; with reference to hydrogen ion concentration, and 
probably other inhibiting factors, they are unstable and continually 
moving toward an equilibrium which, so far as we know, does not 
coincide with the equilibrium obtaining in the plant. 

Catalase 

Determinations of catalase activity (table XX) were made on 
12 samples of bark, of which nos. 9 and 10 form a set from one 
tree and nos. 13 to 20 a set from another tree. Nos. 3 and 4 each 
came from different trees and are the ones used for most of the 
oxidase work reported in this paper. They were about i year old 
when tested for catalase. The other samples were freshly prepared 
for this work in December 191 7 and January 1918. The limbs 
from which they came were carefully cleaned to remove lichens, 



iqiq] 



ROSE— BUSTER CANKER 



'^iZ 



Plenrococcus, etc., since microchemical work had shown that such 
growths have a high catalase activity. The bark was then shaved 
off, ground in a meat chopper, and allowed to dry on filter paper 
at room temperature. In the case of samples 9, 10, 14, 16, 18, and 
20, calcium carbonate was added during the grinding process at 
the rate of 0.5 gm. to each 10 gm. of unground bark, to prevent 
destruction of catalase by the acids of the bark (2) or of the hydro- 
gen peroxide used. The dried bark was finally ground to a powder 
and only that part used which passed through an 80-mesh sieve. 



TABLE XX 
Catalase activity of apple bark 



Sample 

NUMBER 



3 

4 
9 

10 
13 

14 

IS 

16 

17 
18 

19 

20 



Description of sample 



Healthy, from sound limb, no car- 
bonate 

Diseased, no carbonate 

Healthy, from sound limb, plus car- 
bonate 

Diseased, plus carbonate 

Healthy, from sound limb, no car- 
bonate 

Healthy, from sound limb, plus car- 
bonate 

Healthy ( ?) 5 cm. from canker, no 
carbonate 

Healthy ( ?) 5 cm. from canker, plus 
carbonate 

Diseased, no carbonate 

Diseased, plus carbonate 

Dead, no carbonate 

Dead, plus carbonate 



Tempera- 
ture 



23-5 



22.0 



20.5 
22.5 



Positive pressure in cm. 



After s min. After lo min. 



0.55 

0.83 
S-49 

0.52 

1.83 

0.54 

0-73 
0-5S 
1-47 
5 00 
7.01 



0. ID 

0-95 

1 . 26 
8.59 

o. 70 

3.02 

0.65 

1 .01 
0.81 
2.74 

7-37 
12.17 



Tests were made at room temperature by means of the simpli- 
fied Bunzell apparatus, using 0.03 or o.iogm. of bark powder, 
I cc. of water, and 4 cc. of 25 per cent hydrogen peroxide. After 
the experiment was set up the apparatus was allowed to stand for 
half an hour, when the manometers were closed and the solutions 
mixed. The apparatus was shaken for 10 seconds at the end of 
each minute and readings taken after 5 and 10 minutes. All tests 
were made in duphcate or quadrupHcate, a water blank being 
included for temperature corrections as in the oxidase work. 



134 BOTANICAL GAZETTE [February 

A test for catalase was run also on the fungus powder pre- 
viously mentioned, using 0.03 gm. in each tube and calculating 
the results to the basis of o . 10 gm. The average mercury rise 
(positive pressure) produced in 3 tubes was i .65 cm. in 5 minutes 
and 2.57 cm. in 10 minutes, or, calculated to the basis of o . 10 gm., 
5.49 cm. in 5 minutes, and 8.55 cm. in 10 minutes. It is worthy 
of note that a powder prepared from Nummularia mycelium grown 
in Raulin's solution, which is acid to litmus, showed no catalase 
activity. Experiments with different amounts of material showed 
that the positive pressure varies directly with the amount of 
material used. It was deemed legitimate, therefore, to calculate 
all results to the basis of o . 10 gm. of bark powder, and the figures 
for final tabulation were so calculated. 

The results for samples 14, 16, 18, all from the same tree, show 
that diseased bark (sample 18) had more than twice the catalase 
activity of seemingly healthy bark 5 cm. away from the canker 
(sample 16), but only nine-tenths of that of bark from a sound 
unaffected limb on the same tree (sample 14). Dead cankered 
bark from this tree (sample 20) had 4 times the catalase activity 
of healthy bark, 12 times that of seemingly healthy bark next the 
canker, and nearly 5 times that of diseased bark. In the case of 
samples 9 (healthy) and 10 (diseased), the results are reversed, 
since the diseased had a catalase activity nearly 7 times greater 
than that of the healthy bark. The reason for the discrepancy 
between these two sets is not clear. The high catalase activity 
of sample no. 10 can hardly have been due to the presence of 
lichens, etc., or of an admixture of really dead bark, for precautions 
were taken when the samples were removed to avoid these sources 
of error. From the present data the only conclusion that can be 
drawn is that diseased bark from different trees varies considerably 
in its catalase activity, and that in general the more completely 
the bark is destroyed by the fungus the greater is its catalase 
activity. This condition is probably to be explained by the 
presence in the diseased bark of considerable amounts of mycelium 
which, as shown, produces a catalase of its own. 

The seemingly healthy bark near the canker when compared 
with sound and with diseased bark appears to form an exception 



iqiq] 



ROSE— BLISTER CANKER 



135 



in the series. Its catalase activity is less than that of either of the 
others and seems to be less affected by tissue acids when no car- 
bonate is added. It is possible that near the canker the host's 
catalase is injured by materials from the fungus, even in advance 
of actual invasion by the hyphae. The fungus catalase may not 
appear here at all, but only later in the diseased bark, and in 
increasing amounts as the amount of mycehum increases. 

The oxidase activity of samples 13, 15, 17, 19, together with the 
catalase activity of samples 14, 16, 18, 20, identical with them 
except for the addition of carbonate, are. given in table XXI. 

TABLE XXI 

Catalase activity of apple bark 



Description of sample 


Manometer readings expressed 

in cm. of mercury usi.vg . i 

gm. of bark povvtjer 




Catalase 


Oxidase 


Healthy 


3.02 

1 .01 

2.50 

12.17 


I 16 


Healthy ( ?) 5 cm. from canker 

Diseased 

Dead 


1-47 
1-95 
0.88 



It will be seen that there is a gradual increase in oxidase activ- 
ity from healthy to diseased bark, but a marked decrease in the 
case of dead bark. The catalase is considerably lower in apparently 
healthy bark near a canker than in the bark of an unaffected limb, 
but very much higher in the bark killed by the fungus than in 
bark from a healthy hmb. 



Microchemical analysis 

Tests for oxidase, peroxidase, and catalase were made on fresh 
bark, all others on bark preserved in 50 per cent alcohol. The 
results are given in table XXII. 

In making the tests for oxidase (direct action) and peroxidase 
(indirect action), the brownish purple color due to oxidation of 
benzidine was found most marked at first, in both healthy and 
diseased bark, in a zone 2 or 3 cells wide just inside the cork and 
in the pith rays. Later it came to about the same intensity over 



136 



BOTANICAL GAZETTE 



FEBRUARY 



the whole section. Catalase, judging by evolution of gas when 
H2O2 was added, was evenly distributed in all the tissues. Tests 
with FeCl^ on sections of bark successively farther and farther 

TABLE XXII 

Results of microchemical tests on healthy and diseased bark 



Substance 



Cellulose 

Pectin. . 

Lignin. . 

Tannin. . 
Nitrates . 

Fats. . . . 



Calcium (crystals) , 



Calcium oxalate (crys- 
tals) 

Direct reducing sugars 

Starch 

[Cyanogenic gluco- 
j side, probably 

[ amygdalin 

Oxidase (direct action) 

Peroxidase (indirect 
action) 

Catalase 



per 



IKI and 75 

cent H2SO4 
Ruthenium red 
Phloroglucin and 

HCl 
Ferric chloride 
Diphenylamine in 

75 per cent H2SO4 
Sudan III 



50 per cent acetic 

acid 
50 per cent H2SO4 

Oxalic acid 

Fliickigers reagent 

IKI 

Picric acid and 

Na.CO^ 
Berlin blue reaction 
I per cent benzidine 

in 50 per cent 

alcohol 

I per cent benzidine" 

and H2O2 
H2O2 



Reaction 



Healthy 



Diseased 



+ + 
+ 

+ in bast 

+ + 



+ Especially in 
parenchyma next 
to cork 
+ + Crystals sol- 
uble 
+ -f CaS04 formed 
+ -f Crystals not 
changed 
+ 
+ + 
+ 

+ 
+ 



+ 
+ 



+ 
+ 

+ in bast 

+ 



+ Same as for 
healthy bark 

-\--\- Crystals sol- 
uble 
-f+ CaS04 formed 
-{--\- Crystals not 
changed 
+ + 
+ 



+ 

+ 
+ 



distant from the badly browned region showed steadily increasing 
amounts of tannin. Pectin seemed to be present in about equal 
amounts in both healthy and diseased tissues. 

Macrochemical analysis 

Six samples were analyzed. The analytical methods used for 
4 of them are based on those employed by Koch for the quantitative 
study of animal and plant tissues (21, pp. 199-207). The differ- 
ence in material required minor variations from these methods, 
but it is not thought necessary to describe them here. The other 



1919] ROSE— BUSTER CANKER 137 

2 were analyzed according to a method devised by Kraybill 
(unpublished work) in a study of the chemical composition of tomato 
plants. Material for 4 of the samples, healthy i and 2 and' diseased 
I and 2, was taken from 8-10 cm. apple limbs cut in January at 
the Missouri State Fruit Experiment Station, and shipped from 
there by express. As soon as these samples arrived they were pre- 
pared as follows: bark designated as "healthy" was removed from 
sound limbs with a box scraper and cut into pieces half an inch 
square; about 150 gm. were then weighed quickly on a torsion bal- 
ance to hundredths of a gram and put into enough redistilled alcohol 
(95 per cent) to give an alcohol concentration of approximately 
85 per cent. The bottles containing the samples were then set 
into a steam bath until the alcohol came to a boil, then on top for 
I hour longer, to inactivate the enzymes. 

Bark designated as "diseased" was taken from 8-10 cm. 
limbs showing well developed but not old cankers, usually about 
45 cm. long. A strip of moist browned bark 2-3 cm. wide around 
the outside of the canker was removed with the box scraper, 
cut up, weighed, and preserved as described. This material usually 
contained small portions of the seemingly healthy bark outside 
of the canker, but never any part of the black dead material that 
often covers the central part of the cankered areas. Healthy 
samples 3 and 4 were taken from a 7 cm. Hmb cut in April when 
the bark peeled easily, to avoid removing small shavings of wood 
along with the bark, as was inadvertently done in the case of healthy 
samples i and 2 (see discussion of table XXV) . Healthy samples 3 
and 4 were not extracted with hot alcohol and ether as in the 
method described by Harvey (21); instead the alcohol for pre- 
serving was filtered into a 1000 cc. flask and made up to volume. 
One-twentieth ahquots were then pipetted off into small beakers, 
evaporated to a syrup, and used later for dry weight and other 
estimations. The partly extracted bark was dried as described 
for the other samples, weighed, ground, allowed to come to air dry 
condition, and one-twentieth ahquots weighed out as before. 
This method of handling the material is much shorter than the 
Koch method and is very satisfactory if one is not interested in the 
distribution of substances in the various fractions. 



138 BOTANICAL GAZETTE [February 

Dry weight. — One-tenth or one-twentieth ahquots, in tared 
crucibles or beakers, were brought to constant weight in a vacuum 
desiccat(5r after intermittent drying for various lengths of time at 
about 100° C. 

Nitrogen. — Estimations were made by the Kjeldahl- Gunning 
method, modified to include the nitrogen of nitrates. For healthy 
samples i and 2 and diseased samples i and 2 estimations were 
made separately on fractions 2 and 3; no nitrogen was found in 
fraction 2. Estimations for healthy samples 3 and 4 were made 
on one-twentieth of the alcohol extract combined with one-twentieth 
of the partly extracted bark. 

Carbohydrates. — Healthy samples i and 2, diseased samples i 
and 2 : in the case of fraction 2, direct reducing sugars, and reducing 
sugars after mild hydrolysis, were estimated by the Bertrand 
volumetric method and calculated as dextrose by use of the Munson 
and Walker tables (34) . The more important details of manipula- 
tion, including precipitation of non-sugars, are given by Cul- 
pepper, Foster, and Caldwell (16). The polysaccharides in 
fraction 3 were estimated as dextrose, but after 2 . 5 instead of 
5 hours' hydrolysis (16). 

Healthy samples 3 and 4: one-twentieth of the air dry, partly 
extracted bark was further extracted on a filter with about 200 cc. 
of water at 40° C, the filtrate being collected in a beaker containing 
one-twentieth of the alcohol extract. Estimation of sugars and 
polysaccharides in the combined extracts were then made as 
already described. The results of the analysis are given in tables 
XXIII and XXIV and summarized in table XXV. 

The most important differences shown in the tables, as between 
healthy samples i and 2 and diseased samples i and 2, are as fol- 
lows : diseased tissue contains 3 . 23 per cent more dry matter than 
healthy, although here much depends on the manner in which the 
sample is taken; on the basis of dry weight, fraction i is larger 
in the diseased by 4.56 per cent (nearly doubled), indicating a 
synthesis of lipoids by the fungus; fraction 3, the alcohol- water- 
insoluble residue, is larger by i .83 per cent, while fraction 2, con- 
taining the alcohol-water-soluble substances, is smaller by 6 . 27 per 
cent. These results are strikingly similar to those found by 



I9I9] 



ROSE— B LIS TER CA NKER 



139 



Culpepper, Foster, and Caldwell (16), working with black rot 
of apples, caused by Sphaeropsis malontm. The increase in total 

TABLE XXIII 
Results of analysis of healthy bark 



Material 


Percentage wet 
weight 


Percentage wet 
weight 


Percentage dry 
weight 


Percentage dry 
weight 


Total solids 


Sample i 

51-55 

2.56 

14.84 

34-14 
0.23 
1-58 

0-55 

1.07 

7.40 

8-47 
Sample 3 

46.13 
0.217 

0-915 
0.634 

7-524 


Sample 2 

51-50 

2-59 

13.26 

35-45 
0.23 
1 .60 

0.54 

0. 22 

7-56 

7-78 

Sample 4 
46.24 
0.231 
0.949 

0.662 

7.189 


Sample i 


Sample 2 


" Fi 


4-97 
28.79 
66.21 

0.45 
3.06 

1.07 

2.08 

14-35 

16.43 
Sample 3 





" F, 


5 04 

25-85 

69.08 

0.46 

3-13 

1-05 
I. 71 

14-74 

16.45 

Sample 4 


" F3 


Total nitrogen 


Direct reducing sugars 

Reducing sugars after mild 

hydrolysis 

Reducing sugars after strong 

hydrolysis Fi and F2 

Reducing sugars after strong 

hydrolysis F3 

Reducing sugars after strong 

hydrolysis, total 

Total solids 


Total nitrogen 

Direct reducing sugars 

Reducing sugars after mild 
hydrolysis 


0.46 
1.96 

1.72 

16.20 


0.50 
2.05 

1-45 
16.55 


Reducing sugars after strong 
hydrolysis 





TABLE XXIV 

Results of analysis of diseased bark 



Material 


Sample i 
Percentage 
wet weight 


Sample 2 
Percentage 
wet weight 


Sample 1 Sample 2 
Percentage Percentage 
dry weight dry weight 


Total solids 


54-29 

4-69 

11.52 

38.07 

0-45 
1.42 

0.66 

0.13 

8.80 

8-93 


55-12 

5-77 
11.52 

37-94 
0.45 
1.56 

0.59 

0.38 

8.84 

9.22 






" Fi 


8.64 

21 .21 

70.16 

0.83 

2.62 

1.22 

0.23 

16.21 

16.44 


10.47 
20.89 

68 80 


" F2 


" F3 


Total nitrogen 


81 


Direct reducing sugars 

Reducing sugars after mild 
hydrolysis 


2.83 

1.07 

0.70 

16.04 

16.74 


Reducing sugars after strong 
hydrolysis Fi and F2 

Reducing sugars after strong 
hydrolysis F3 

Reducing sugars after strong 
hydrolysis, total 



140 



BOTANICAL GAZETTE 



FEBRUARY 



nitrogen in diseased bark may be due to fixation by the fungus 
or to a withdrawal of nitrogen from the surrounding tissue. Fur- 
ther data are necessary before a conclusion can be reached. Cul- 
pepper, Foster, and Caldwell found protein-nitrogen content 
of fraction 2 for diseased apples larger than for normal ones, but 
the total nitrogen for the whole tissue smaller for the former than 
for the latter. 

TABLE XXV 

Summary 





Average percentage wet weight 


Average percentage dry weight 


Material 


Healthy i Healthy 3 

and 2 i and 4 


Diseased i 

and 2 


Healthy i 

and 2 


Healthy 3 
and 4 


Diseased i 
and 2 


Total solids 


51-53 

2.58 

14-05 

34.00 

0.23 

1-59 

0-55 
8.12 


46.19 

0.24 
0.93 

0.65 
7-35 


54-70 

5 23 

11.52 

37-57 

0.45 

1.49 

0.62 
9.08 








*' Fx 


5.00 

27-32 

67.65 

0.46 

3.00 

1.06 

16.44 




0.48 
2.00 

1-59 
16.38 


9.56 


" F2 


21.05 


" F, 


69.48 


Total nitrogen 


0.82 


Direct reducing sugars .... 
Direct reducing sugars after 

mild hydrolysis 

Direct reducing sugars after 

strong hydrolysis 


2-73 

I-15 

16.59 



Results with healthy samples 3 and 4 furnish little of additional 
interest. They show, however, that as far as total nitrogen and 
starch are concerned, the small amount of wood in the other 2 
healthy samples had no effect on the results. The difference in 
the case of dry weight and reducing sugars before and after hy- 
drolysis is probably due to the fact that samples 3 and 4 were taken 
from a limb cut early in the growing season, while samples i and 2 
were taken from hmbs cut in the dead of winter. 



Estimation of tannin 

The method used was that of Lowenthal, as modified by 
Proctor (34, p. 150). Material for analysis was taken from 
8-12 cm. Ben Davis limbs cut in November, December, and 
January. The bark was cut off as already described, ground in a 
meat grinder, and transferred to a glass moist chamber at once. 
About 10 gm. were then weighed out and set to boil in 400 cc. of 



iQig] 



ROSE— BLISTER CANKER 



141 



water as required by the Lowenthal method; at the same time 
duplicate samples were taken for moisture determination. What- 
ever may have been the errors introduced by this method, the 
agreement between duplicates taken for moisture determination 
was very close in most cases, as is shown in table XXVI. 

TABLE XXVI 

Percentage of dry matter in duplicate samples of 
various lots of bark analyzed 



Sample 

Healthy i . . . 

2... 

" 3.-- 
Diseased 4 . . . 

" s-.. 

6... 

Dead 7 

" 8 

" 9 



Duplicate i 



Duplicate 2 



Average 



The results of the analysis of 9 different samples of bark are 
shown in table XXVII. 

TABLE XXVII 

Percentage of tannin in healthy and diseased 
APPLE bark 



Description of sample 


Tannin (percent- 
age dry weight) 


Healthy 

I 


5. 16 


2 8-10 cm. from canker 


3.64 


3. From same tree as 6 and 9 

Average 


3.38 
4.06 


Diseased 


2.49 




3.56 


6 


2.93 


Average 


2 .99 


Dead (from surface of canker) 


0. 25 


8 


I. 14 





I. 51 


Average 


0.97 







The Lowenthal method probably determines merely the 
easily water-soluble tannins, but fails to reach those tied up with 



142 BOTANICAL GAZETTE [February 

the suberin. If suberin is for any reason more abundant in the 
diseased bark, an error would thus be introduced which might 
invalidate any comparisons based on the results obtained. Sub- 
ject to this possible correction the results shown in table XXVII 
confirm those obtained in the microchemical analysis; that is, 
they show a progressive decrease in tannin as the bark is more and 
more affected by the disease. Healthy bark was found to contain 
on the average 4.06 per cent of tannin, diseased 2.99, and dead 
o .97. If sample i healthy, which gave a high figure, and sample 7 
dead, which gave a low figure, be eliminated, the averages become 
healthy 3.51, diseased 2.99, dead 1.33. The figures for samples 
3, 6, and 9, all from the same tree, are healthy 3 .38, diseased 2 .93, 
dead i .51. There is undoubtedly a difference between bark from 
a sound limb and seemingly healthy bark from a limb that is badly 
cankered. The latter is usually shghtly browned throughout 
when first cut off and rapidly becomes reddish brown on exposure 
to air. Really healthy bark under such conditions shows only a 
slight browning. 

Whatever the results with apple bark may mean, they are not 
in agreement with the statement made by Kerr (see Cook and 
Wilson, 15, p. 26, footnote) that because of the greater stability 
of tannin and the disappearance of other constituents "all decayed 
wood and bark give higher tannin contents, no matter what causes 
the decay." If confirmed by further analyses they would indicate 
a different relation between host and parasite with reference to 
tannin in the case of blister canker than obtained in any of the 
cases studied by Kerr. Leaching of tannin may account for 
the low percentage found in dead apple bark, as suggested by the 
chemist of the Chestnut Tree Blight Commission (15, p. 6) for old 
cankers of chestnut blight, but can hardly be responsible for the 
condition found in diseased bark. 

Summary 

I. Measurements with the simplified Bunzell apparatus show 
that apple bark attacked by Nummnlaria discreta causes about 
twice as much oxidation of pyrogallol, pyrocatechin, guaiacol, and 
benzidine as does healthy bark. 



1919] ROSE— BLISTER CANKER 



143 



2. The gradual slowing down of oxidation in the Bunzell 
apparatus is shown to be due, in part at least, to increasing hydrogen 
ion concentration, brought about by the oxidation process itself. 
The equilibrium reached in the oxidase apparatus seems to be a 
false one, which can be disturbed by the addition of either fresh 
oxidase reagent or plant material. When tested by the formula 
for a unimolecular reaction, the oxidase reaction gives values for 
k, which indicate clearly a Hnear relationship between time and 
amount of change and suggest that the oxidase is a catalytic agent. 

3. The hydrogen ion concentration of diseased bark (P3 = 5 .61) 
is definitely less than that of healthy bark (Pjj = 5.i5). Work 
with buffer solutions shows that this difference is not great enough 
to account for all of the difference in the oxidase activity of the 
two kinds of material. When mixtures of the two are brought to 
the same hydrogen ion concentration by means of buffer solutions, 
diseased bark still shows greater oxidase activity. 

4. The temperature and duration of drying have an eifect on 
the acidity and the oxidase activity of both healthy and diseased 
bark. 

5. Eight-tenths per cent gelatine increases the oxidase activity 
of both kinds of bark. This may be due to the action of gelatine 
as a protective colloid which prevents precipitation of the "oxidase." 
It is not due to buffer action. 

6. The concentration of hydrogen ion necessary or complete 
inhibition of oxidase activity of healthy bark lies between 3 .55 and 
3 .80X10"'^; for that of diseased bark between 3 .55 and4.27Xio~^ 

7. Oxidation in the apparatus comes to an end only after several 
days instead of after a few hours, as stated by Bunzell. 

8. When the "oxidase" is precipitated in 2 fractions, the first 
has greater oxidizing power than the second, and the 2 combined 
have slightly greater oxidizing power than when tested separately. 

9. Catalase determinations gave the following results: healthy 
3.02 (cm. positive pressure), seemingly healthy 5cm. from the 
canker i.oi, diseased 2.74, dead 12.17; results from oxidase 
determinations for the same stages were 1.16, 1.47, i-95, 1.85 
(cm. negative pressure). These results show some discrepancies, 
but justify the general statement that the more severely the bark 



144 BOTANICAL GAZETTE [February 

is attacked by the fungus the greater is its catalase activity, and 
that catalase activity in part is in indirect ratio to oxidase activity. 

10. Microchemical tests indicate, for diseased bark, a partial 
disintegration of cellulose, a disappearance of cyanogenic glucoside, 
and a lower content of starch, calcium oxalate, and tannins. 

11. Macrochemical analyses show that diseased bark has a 
higher percentage of dry matter, lipoids, alcohol-water-insoluble 
residue, and total nitrogen, but a lower percentage of alcohol- water- 
soluble material than healthy bark. The percentage of carbo- 
hydrates in both tissues seems to be about the same. Differences 
in tannin content are definite but not large. Sound healthy bark 
contains more than diseased bark and diseased bark more than 
dead bark from the surface of the canker. 

12. The greater oxidase activity of diseased bark is probably 
due to the combined activity of the oxidases of fungus and host, 
lower acidity, and possibly to a greater degree of dispersion of the 
oxidizing agent. The lower tannin content of diseased bark may 
also be a contributing factor. 

The writer wishes to acknowledge his indebtedness to 
Dr. William Crocker, Dr. F. C. Koch, and Dr. Sophia H. 
EcKERSON for valuable suggestions and criticism during the course 
of the investigation. Thanks are due to Dr. Paul Evans of the 
Missouri State Fruit Experiment Station for bark material used 
in the experiments. 

U.S. Department of Agriculture 
Washington, D.C. 

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